This proposal outlines a plan to investigate the mechanism of assembly of E. coli ribosomes both in vitro and in vivo. The ribosome is the macromolecular machine responsible for all protein synthesis in cells, and it is efficiently assembled from over 50 components. Using a combination of biophysical methods, we will investigated the order of events in assembly and the kinetics of assembly in vitro. We will examine the role of assembly cofactors in E. coli, and examine the effects of antibiotics on the assembly process. We have developed a novel isotope pulse-chase assay that enables measurement of binding kinetics for ribosomal proteins using mass spectrometry. We are developing a two-photon fluorescence correlation microscope to monitor the assembly of fluorescently labeled ribosomal proteins in real time. Finally, we are extending our mass spectrometry analysis to isotope pulse experiments in living bacteria, which allows us to directly monitor the biogenesis of ribosomes. Taken together, these studies will provide new mechanistic insights into the critical process of ribosome assembly.7. Project Narrative The assembly of ribosomes is essential for protein synthesis in all cells. The proposed studies will help elucidate the understanding of ribosome biogenesis in bacteria. Resulting insights may lead to novel anti- infective strategies.